Immunogenic composition

ABSTRACT

The present application discloses an immunogenic composition comprising a Hib saccharide conjugate, at least one additional bacterial, for example  N. meningitidis , saccharide conjugate(s), and a further antigen selected from the group consisting of whole cell pertussis and hepatitis B surface antigen, wherein the saccharide dose of the Hib saccharide conjugate is less than 5 μg.

This application is a continuation application of U.S. application Ser.No. 13/778,633, filed Feb. 27, 2013 (to issue as U.S. Pat. No. 8,883,163on Nov. 14, 2014), which is a continuation application of U.S.application Ser. No. 11/917,726, filed Dec. 14, 2007 (now U.S. Pat. No.8,398,983, issued Mar. 19, 3013), pursuant to 35 U.S.C. §371 as a UnitedStates National Phase Application of International Patent ApplicationSerial No. PCT/EP2006/006220 filed Jun. 23, 2006, which claims priorityfrom Great Britain Application No. 0513069.5 filed in the United Kingdomon Jun. 27, 2005, from Great Britain Application No. 0513071.1 filed inthe United Kingdom on Jun. 27, 2005, from Great Britain Application No.0515556.9 filed in the United Kingdom on Jul. 28, 2005, from GreatBritain Application No. 0524204.5 filed in the United Kingdom on Nov.28, 2005, from Great Britain Application No. 0526040.1 filed in theUnited Kingdom on Dec. 21, 2005, and from Great Britain Application No.0526041.9 filed in the United Kingdom on Dec. 21, 2005, the entirecontents of each of which are hereby incorporated by reference.

The present application relates to immunogenic compositions and vaccinescomprising a Hib saccharide conjugate and at least one additionalbacterial saccharide conjugate(s) combined with further antigen(s)including whole cell pertussis and/or hepatitis B surface antigen,processes for making such immunogenic compositions and vaccines, anduses and methods of immunisation using the immunogenic composition andvaccine.

Bacterial polysaccharides have been shown to be effective immunogens foruse in vaccines, particularly when conjugated to a carrier protein.Commercial conjugate vaccines are available against Haemophilusinfluenzae type b (Hibtiter® Wyeth-Lederle), pneumococcalpolysaccharides (Prevnar®—Wyeth-Lederle) and meningococcalpolysaccharides (Meningitec®—Wyeth-Lederle and Menactra®—Sanofi).

Immunogenic compositions and vaccines comprising a Hib conjugate, a N.meningitidis saccharide conjugate, whole cell pertussis and hepatitis Bsurface antigen have also been described. For instance WO 02/00249discloses a seven valent immunogenic composition comprising a diphtheriatoxoid, tetanus toxoid, whole cell pertussis, Hepatitis B surfaceantigen, Hib conjugate, MenA conjugate and MenC conjugate. The clinicaltrial results presented use a 5 μg saccharide dose of each of thebacterial saccharide conjugates.

The present invention concerns the provision of a combination vaccinecomprising a Hib conjugate, an additional bacterial saccharide conjugate(for example an N. meningitidis saccharide conjugate) and furtherantigens including one or both of whole cell pertussis and Hepatitis Bsurface antigen. The saccharide doses used allow a good immune responseto be generated against Hib and the additional bacterial saccharides aswell as against the pertussis component and/or the Hepatitis B surfaceantigen component.

The inventors have surprisingly found that by reducing the saccharidedose of Hib and/or additional bacterial conjugates to below 5 μg perdose, in a combination vaccine comprising whole cell pertussis and/orHepatitis B surface antigen, a good immune response to the saccharideconjugates is maintained and the immunogenicity of whole cell pertussisand Hepatitis B surface antigen is enhanced (for example such that theGMC for Pw and/or Hepatitis B as measured by ELISA is higher than thelevel achieved after immunisation with an immunogenic compositioncontaining a 5 μg saccharide dose of each conjugate).

Accordingly, a first aspect of the invention provides an immunogeniccomposition comprising a Hib saccharide conjugate, at least oneadditional bacterial saccharide conjugate(s), and a further antigenselected from the group consisting of whole cell pertussis and hepatitisB surface antigen, wherein the saccharide dose of the Hib saccharideconjugate is less than 5 μg per dose.

DETAILED DESCRIPTION

The immunogenic composition of the invention comprises a Hib saccharideconjugate, and/or at least or exactly one, two, three or four bacterialsaccharide conjugates, for example N. meningitidis saccharideconjugate(s), and a further antigen selected from the group consistingof whole cell pertussis and hepatitis B surface antigen, wherein thesaccharide dose of the Hib saccharide conjugate is less than 5 μg orless than 4 μg, or between 1-4 μg or 1-3 μg, or 2-4 μg or 2-3 μg oraround or exactly 2.5 μg and optionally the saccharide dose of the oreach of the at least or exactly one, two, three or four additionalbacterial saccharide conjugate(s) (for example N. meningitidissaccharide conjugate(s)) is less than 1-4 μg, 9 μg, 8 μg, 7 μg, 6 μg, 5μg or 4 μg, or between 1-10 μg, 1-8 μg, 1-6 μg, 1-5 μg, 1-4 μg, 1-3 μg,or 2-4 μg or 2-3 μg or about or exactly 2.5 μg.

The term “saccharide” includes polysaccharides or oligosaccharides.Polysaccharides are isolated from bacteria or isolated from bacteria andsized to some degree by known methods (see for example EP497524 andEP497525) and optionally by microfluidisation. Polysaccharides can besized in order to reduce viscosity in polysaccharide samples and/or toimprove filterability for conjugated products. Oligosaccharides have alow number of repeat units (typically 5-30 repeat units) and aretypically hydrolysed polysaccharides.

The “saccharide dose” is measured in the amount of immunogeniccomposition or vaccine that is administered to a human.

A Hib saccharide is the polyribosyl phosphate (PRP) capsularpolysaccharide or oligosaccharide of Haemophilus influenzae type b.

“At least one additional bacterial saccharide conjugate(s)” refers to aconjugate of any bacterial saccharide linked to a carrier protein. Thebacterial saccharide is optionally a capsular saccharide derived from anN. meningitidis strain, a S. pneumoniae strain, an S. typhi strain, orany of the bacterial saccharides described herein.

“At least one N. meningitidis saccharide conjugate(s)” refers to N.meningitidis serogroup A capsular saccharide (MenA), N. meningitidisserogroup C capsular saccharide (MenC), N. meningitidis serogroup W135capsular saccharide (MenW), to N. meningitidis serogroup Y capsularsaccharide (MenY), N. meningitidis serogroup B capsular saccharide(MenB), serogroup C and Y capsular saccharides (MenCY), serogroup C andA capsular saccharides (MenAC), serogroup C and W135 capsularsaccharides (MenCW), serogroup A and Y capsular saccharide (MenAY),serogroup A and W135 capsular saccharides (MenAW), serogroup W135 and Ycapsular saccharides (Men WY), serogroup A, C and W135 capsularsaccharide (MenACW), serogroup A, C and Y capsular saccharides (MenACY);serogroup A, W135 and Y capsular saccharides (MenAWY), serogroup C, W135and Y capsular saccharides (MenCWY); or serogroup A, C, W135 and Ycapsular saccharides (MenACWY), serogroup B and C capsular saccharides(MenBC), serogroup B, C and Y capsular saccharides (MenBCY), serogroupB, C and A capsular saccharides (MenABC), serogroup B, C and W capsularsaccharides (MenBCW), serogroup A, B and Y capsular saccharide (MenABY),serogroup A, B and W capsular saccharides (MenABW), serogroup B, W135and Y capsular saccharides (MenBWY), serogroup A, B, C and W135 capsularsaccharide (MenABCW), serogroup A, B, C and Y capsular saccharides(MenABCY); serogroup A, B, W135 and Y capsular saccharides (MenABWY),serogroup B, C, W135 and Y capsular saccharides (MenBCWY); or serogroupA, B, C, W135 and Y capsular saccharides (MenABCWY).

For example, any of the combinations of N. meningitidis saccharideconjugates listed above, with or without the addition of Hib saccharideconjugate, may be present at a saccharide dose of less than 5 μg or lessthan 4 μg, or 1-4 μg or 1-3 μg, or 2-4 μg or 2-3 μg or around or exactly2.5 μg.

“Around” or “approximately” are defined as within 10% more or less ofthe given figure for the purposes of the invention.

In an embodiment the saccharide dose of Hib can be the same as, morethan or less than the saccharide dose of the N. meningitidis saccharideconjugate. The saccharide dose of the Hib saccharide conjugate is forexample 100% or less than 90%, 80%, 75%, 70%, 60%, 50%, 40%, 30%, 20% or10% of the mean or lowest saccharide dose of the at least one additionalbacterial (for example N. meningitidis) saccharide conjugate(s). Thesaccharide dose of the Hib saccharide is for example between 20% and60%, 30% and 60%, 40% and 60% or around or exactly 50% of the mean orlowest saccharide dose of the at least one additional bacterial (forexample N. meningitidis) saccharide conjugate(s).

In an embodiment of the invention, the dose of the or each of the atleast one additional bacterial (for example N. meningitidis) saccharidesis the same, or approximately the same.

Examples of immunogenic compositions of the invention are compositionsconsisting of or comprising:

Examples of immunogenic compositions of the invention are compositionsconsisting of or comprising:

Hib conjugate and MenA conjugate and MenC conjugate, optionally atsaccharide dose ratios of 1:2:2, 1:2:1, 1:4:2, 1:6:3, 1:3:3, 1:4:4,1:5:5, 1:6:6 (w/w). Optionally, the saccharide dose of MenA is greaterthan the saccharide dose of MenC.

Hib conjugate and MenC conjugate and MenY conjugate, optionally atsaccharide dose ratios of 1:2:2, 1:2:1, 1:4:2, 1:4:1, 1:8;4, 1:6:3,1:3:3, 1:4:4, 1:5:5, 1:6:6 (w/w). Optionally, the saccharide dose ofMenC is greater than the saccharide dose of MenY.

Hib conjugate and MenC conjugate and MenW conjugate, optionally atsaccharide dose ratios of 1:2:2, 1:2:1, 1:4:2, 1:4:1, 1:8;4, 1:6:3,1:3:3, 1:4:4, 1:5:5, 1:6:6 (w/w). Optionally the saccharide dose of MenCis greater than the saccharide dose of MenW.

Hib conjugate and MenA conjugate and MenW conjugate, optionally atsaccharide dose ratios of 1:2:2, 1:2:1, 1:4:2, 1:4:1, 1:8;4, 1:6:3,1:3:3, 1:4:4, 1:5:5, 1:6:6 (w/w). Optionally, the saccharide dose ofMenA is greater than the saccharide dose of MenW.

Hib conjugate and MenA conjugate and MenY conjugate, optionally atsaccharide dose ratios of 1:2:2, 1:2:1, 1:4:2, 1:4:1, 1:8:4, 1:6:3,1:3:3, 1:4:4, 1:5:5, 1:6:6 (w/w). Optionally the saccharide dose of MenAis greater than the saccharide dose of MenY.

Hib conjugate and MenW conjugate and MenY conjugate, optionally atsaccharide dose ratios of 1:2:2, 1:2:1, 1:1:2, 1:4:2, 1:2:4, 1:4:1,1:1:4, 1:3;6, 1:1:3, 1:6:3, 1:3:3, 1:4:4, 1:5:5, 1:6:6 (w/w). Optionallythe saccharide dose of MenY is greater than the saccharide dose of MenW.

The “further antigen” comprises one or both of whole cell pertussis (Pw)and Hepatitis B surface antigen (HepB). In an embodiment, the furtherantigen further comprises diphtheria toxoid (DT), tetanus toxoid (TT),acellular pertussis (Pa) and/or polio virus (IPV). In an embodiment, thefurther antigen comprises or consists of DT, TT and Pw. In anembodiment, the further antigen comprises or consists of DT, TT,pertussis antigen (Pa or Pw) and HepB. In an embodiment, the furtherantigen comprises or consists of DT, TT, pertussis antigen (Pa or Pw),HepB and IPV.

Hib and/or the N. meningitidis saccharide(s) included in the immunogeniccompositions of the invention are conjugated to a carrier protein suchas tetanus toxoid, tetanus toxoid fragment C, non-toxic mutants oftetanus toxin, diphtheria toxoid, CRM197, other non-toxic mutants ofdiphtheria toxin [such as CRM176, CRM 197, CRM228, CRM 45 (Uchida et alJ. Biol. Chem. 218; 3838-3844, 1973); CRM 9, CRM 45, CRM102, CRM 103 andCRM107 and other mutations described by Nicholls and Youle inGenetically Engineered Toxins, Ed: Frankel, Maecel Dekker Inc, 1992;deletion or mutation of Glu-148 to Asp, Gln or Ser and/or Ala 158 to Glyand other mutations disclosed in U.S. Pat. No. 4,709,017 or U.S. Pat.No. 4,950,740; mutation of at least one or more residues Lys 516, Lys526, Phe 530 and/or Lys 534 and other mutations disclosed in U.S. Pat.No. 5,917,017 or U.S. Pat. No. 6,455,673; or fragment disclosed in U.S.Pat. No. 5,843,711], pneumococcal pneumolysin, OMPC (meningococcal outermembrane protein—usually extracted from N. meningitidis serogroupB—EP0372501), synthetic peptides (EP0378881, EPO427347), heat shockproteins (WO 93/17712, WO 94/03208), pertussis proteins (WO 98/58668,EPO471177), cytokines, lymphokines, growth factors or hormones (WO91/01146), artificial proteins comprising multiple human CD4+ T cellepitopes from various pathogen derived antigens (Falugi et al (2001) EurJ Immunol 31; 3816-3824) such as N19 protein (Baraldoi et al (2004)Infect Immun 72; 4884-7) pneumococcal surface protein PspA (WO02/091998) pneumolysin (Kuo et al (1995) Infect Immun 63; 2706-13), ironuptake proteins (WO 01/72337), toxin A or B of C. difficile (WO00/61761) or Protein D (EP594610 and WO 00/56360).

In an embodiment, the immunogenic composition of the invention uses thesame carrier protein (independently selected) in the Hib conjugate andthe at least one N. meningitidis saccharide conjugate(s), optionally inthe Hib conjugate and each of the N. meningitidis saccharide conjugates(optionally in all the saccharide conjugates present in the immunogeniccomposition).

In an embodiment, the immunogenic composition optionally comprises a Hibsaccharide conjugate and MenA polysaccharide conjugate, a Hib saccharideconjugate and MenC saccharide conjugate, a Hib saccharide conjugate andMenW saccharide conjugate, a Hib saccharide conjugate and MenYsaccharide conjugate, a Hib saccharide conjugate and MenA and MenCsaccharide conjugates, a Hib saccharide conjugate and MenA and MenWsaccharide conjugates, a Hib saccharide conjugate and MenA and MenYsaccharide conjugates, a Hib saccharide conjugate and MenC and MenWsaccharide conjugates, a Hib saccharide conjugate and MenC and MenYsaccharide conjugates, a Hib saccharide conjugate and MenW and MenYsaccharide conjugates, a Hib saccharide conjugate and MenA, MenC andMenW saccharide conjugates, a Hib saccharide conjugate and MenA, MenCand MenY saccharide conjugates, a Hib saccharide conjugate and MenA,MenW and MenY saccharide conjugates, a Hib saccharide conjugate andMenC, MenW and MenY saccharide conjugates or a Hib saccharide conjugateand MenA, MenC, MenW and MenY saccharide conjugates.

In an embodiment, a single carrier protein may carry more than onesaccharide antigen (WO 04/083251). For example, a single carrier proteinmight be conjugated to Hib and MenA, Hib and MenC, Hib and MenW, Hib andMenY, MenA and MenC, MenA and MenW, MenA and MenY, MenC and MenW, MenCand MenY or Men W and MenY.

In an embodiment, the immunogenic composition of the invention comprisesa Hib saccharide conjugated to a carrier protein selected from the groupconsisting of TT, DT, CRM197, fragment C of TT and protein D.

Where the carrier protein is TT or fragment thereof for Hib and the atleast one N. meningitidis saccharide(s), the total dose of carrier isbetween 2.5-25 μg, 3-20 μg, 4-15 μg, 5-12.5 μg, 15-20 μg, 16-19 μg or17-18 μg.

In an embodiment, the immunogenic composition of the invention comprisesat least one, two, three or four N. meningitidis bacterial saccharide(s)conjugated to a carrier protein selected from the group consisting ofTT, DT, CRM197, fragment C of TT and protein D.

The immunogenic composition of the invention optionally comprises a Hibsaccharide conjugate having a ratio of Hib to carrier protein of between1:5 and 5:1; 1:2 and 2:1; 1:1 and 1:4; 1:2 and 1:3.5; or around orexactly 1:2.5 or 1:3 (w/w).

The immunogenic composition of the invention optionally comprises atleast one meningococcal saccharide (for example MenA and/or MenC and/orMenW and/or MenY) conjugate having a ratio of Men saccharide to carrierprotein of between 1:5 and 5:1, between 1:2 and 5:1, between 1:0.5 and1:2.5 or between 1:1.25 and 1:2.5 (w/w).

The ratio of saccharide to carrier protein (w/w) in a conjugate may bedetermined using the sterilized conjugate. The amount of protein isdetermined using a Lowry assay (for example Lowry et al (1951) J. Biol.Chem. 193, 265-275 or Peterson et al Analytical Biochemistry 100,201-220 (1979)) and the amount of saccharide is determined using ICP-OES(inductively coupled plasma-optical emission spectroscopy) for MenA,DMAP assay for MenC and Resorcinol assay for MenW and MenY (Monsigny etal (1988) Anal. Biochem. 175, 525-530).

In an embodiment, the immunogenic composition of the invention the Hibsaccharide is conjugated to the carrier protein via a linker, forinstance a bifunctional linker. The linker is optionallyheterobifunctional or homobifunctional, having for example a reactiveamino group and a reactive carboxylic acid group, 2 reactive aminogroups or two reactive carboxylic acid groups. The linker has forexample between 4 and 20, 4 and 12, 5 and 10 carbon atoms. A possiblelinker is ADH. Other linkers include B-propionamido (WO 00/10599),nitrophenyl-ethylamine (Geyer et al (1979) Med. Microbiol. Immunol. 165;171-288), haloalkyl halides (U.S. Pat. No. 4,057,685) glycosidiclinkages (U.S. Pat. No. 4,673,574, U.S. Pat. No. 4,808,700) and6-aminocaproic acid (U.S. Pat. No. 4,459,286).

The saccharide conjugates present in the immunogenic compositions of theinvention may be prepared by any known coupling technique. For examplethe saccharide can be coupled via a thioether linkage. The conjugationmethod may rely on activation of the saccharide with1-cyano-4-dimethylamino pyridinium tetrafluoroborate (CDAP) to form acyanate ester. The activated saccharide may thus be coupled directly orvia a spacer (linker) group to an amino group on the carrier protein.Optionally, the cyanate ester is coupled with hexane diamine or ADH andthe amino-derivatised saccharide is conjugated to the carrier proteinusing heteroligation chemistry involving the formation of the thioetherlinkage, or is conjugated to the carrier protein using carbodiimide(e.g. EDAC or EDC) chemistry. Such conjugates are described in PCTpublished application WO 93/15760 Uniformed Services University and WO95/08348 and WO 96/29094.

Other suitable techniques use carbiinides, hydrazides, active esters,norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, TSTU.Many are described in WO 98/42721. Conjugation may involve a carbonyllinker which may be formed by reaction of a free hydroxyl group of thesaccharide with CDI (Bethell et al J. Biol. Chem. 1979, 254; 2572-4,Hearn et al J. Chromatogr. 1981. 218; 509-18) followed by reaction ofwith a protein to form a carbamate linkage. This may involve reductionof the anomeric terminus to a primary hydroxyl group, optionalprotection/deprotection of the primary hydroxyl group’ reaction of theprimary hydroxyl group with CDI to form a CDI carbamate intermediate andcoupling the CDI carbamate intermediate with an amino group on aprotein.

The conjugates can also be prepared by direct reductive aminationmethods as described in U.S. Pat. No. 4,365,170 (Jennings) and U.S. Pat.No. 4,673,574 (Anderson). Other methods are described in EP-0-161-188,EP-208375 and EP-0-477508.

A further method involves the coupling of a cyanogen bromide (or CDAP)activated saccharide derivatised with adipic acid hydrazide (ADH) to theprotein carrier by carbodiimide condensation (Chu C. et al Infect.Immunity, 1983 245 256), for example using EDAC.

In an embodiment, a hydroxyl group on a saccharide is linked to an aminoor carboxylic group on a protein either directly or indirectly (througha linker). Where a linker is present, a hydroxyl group on a saccharideis optionally linked to an amino group on a linker, for example by usingCDAP conjugation. A further amino group in the linker for example ADH)may be conjugated to a carboxylic acid group on a protein, for exampleby using carbodiimide chemistry, for example by using EDAC. In anembodiment, the Hib or at least one N. meningitidis saccharide(s) isconjugated to the linker first before the linker is conjugated to thecarrier protein.

In an embodiment, the Hib saccharide, where present, is conjugated tothe carrier protein using CNBr, or CDAP, or a combination of CDAP andcarbodiimide chemistry (such as EDAC), or a combination of CNBr andcarbodiimide chemistry, (such as) EDAC. Optionally Hib is conjugatedusing CNBr and carbodiimide chemistry (optionally EDAC. For example,CNBr is used to join the saccharide and linker and then carbodiimidechemistry is used to join linker to the protein carrier.

In an embodiment, at least one of the at least one N. meningitidissaccharide(s) is directly conjugated to a carrier protein optionally MenW and/or MenY and/or MenC and/or MenA saccharide(s) is directlyconjugated to a carrier protein. For example MenW; MenY; MenC; MenA;MenW and MenY; MenW and MenC; MenY and MenC; or MenW, MenY and MenC aredirectly linked to the carrier protein. Optionally at least one of theat least one N. meningitidis saccharide(s) is directly conjugated byCDAP. For example MenW; MenY; MenC; MenW and MenY; MenW and MenC; MenYand MenC; or MenW, MenY and MenC are directly linked to the carrierprotein by CDAP (see WO 95/08348 and WO 96/29094).

Optionally the ratio of Men W and/or Y saccharide to carrier protein isbetween 1:0.5 and 1:2 (w/w) or the ratio of MenC saccharide to carrierprotein is between 1:0.5 and 1:2 or 1:1.25 and 1:1.5 or 1:0.5 and 1:1(w/w), especially where these saccharides are directly linked to theprotein, optionally using CDAP.

In an embodiment, at least one N. meningitidis saccharide(s) isconjugated to the carrier protein via a linker, for instance abifunctional linker. The linker is optionally heterobifunctional orhomobifunctional, having for example a reactive amino group and areative carboxylic acid group, 2 reactive amino groups or two reactivecarboxylic acid groups. The linker has for example between 4 and 20, 4and 12, 5 and 10 carbon atoms. A possible linker is ADH.

In an embodiment, MenA; MenC; or MenA and MenC is conjugated to acarrier protein via a linker.

In an embodiment, the N. meningitidis saccharide is conjugated to acarrier protein via a linker using CDAP and EDAC. For example, MenA;MenC; or MenA and MenC are conjugated to a protein via a linker (forexample those with two amino groups at its ends such as ADH) using CDAPand EDAC as described above. For example, CDAP is used to conjugate thesaccharide to a linker and EDAC is used to conjugate the linker to aprotein. Optionally the conjugation via a linker results in a ratio ofsaccharide to carrier protein of of between 1:0.5 and 1:6; 1:1 and 1:5or 1:2 and 1:4, for MenA; MenC; or MenA and MenC.

In an embodiment of the invention, the immunogenic composition comprisesN. meningitidis capsular polysaccharides from at least one, two, threeor four of serogroups A, C, W and Y conjugated to a carrier protein,wherein at least one, two, three or four or each N. meningitidispolysaccharide is either a native polysaccharide or is sized by a factorup to ×1.5, ×2, ×3, ×4, ×5, ×6, ×7, ×8, ×9, ×10 or ×20. For example, theaverage size of at least one, two, three or four or each N. meningitidispolysaccharide is above 50 kDa, 60 kDa, 75 kDa, 100 kDa, 110 kDa, 120kDa or 130 kDa.

“Native polysaccharide” refers to a polysaccharide that has not beensubjected to a process, the purpose of which is to reduce the size ofthe polysaccharide.

“Sized by a factor up to ×2” means that the polysaccharide is subject toa process intended to reduce the size of the polysaccharide but toretain a size more than half the size of the native polysaccharide. ×3,×4 etc. are to be interpreted in the same way i.e. the polysaccharide issubject to a process intended to reduce the size of the polysaccharidebut to retain a size more than a third, a quarter etc. the size of thenative polysaccharide respectively.

In an aspect of the invention, the immunogenic composition comprises N.meningitidis capsular polysaccharides from at least one, two, three orfour of serogroups A, C, W and Y conjugated to a carrier protein,wherein at least one, two, three or four or each N. meningitidispolysaccharide is native polysaccharide.

In an aspect of the invention, the immunogenic composition comprises N.meningitidis capsular polysaccharides from at least one, two, three orfour of serogroups A, C, W and Y conjugated to a carrier protein,wherein at least one, two, three or four or each N. meningitidispolysaccharide is sized by a factor up to ×1.5, ×2, ×3, ×4, ×5, ×6, ×7,×8, ×9 or ×10.

In an embodiment, the mean size of at least one, two, three, four oreach N. meningitidis polysaccharide is between 50 KDa and 1500 kDa, 50kDa and 500 kDa, 50 kDa and 300 KDa, 101 kDa and 1500 kDa, 101 kDa and500 kDa, 101 kDa and 300 kDa as determined by MALLS.

In an embodiment, the MenA saccharide, where present, has a molecularweight of 50-500 kDa, 50-100 kDa, 100-500 kDa, 55-90 KDa, 60-70 kDa or70-80 kDa or 60-80 kDa as determined by MALLS.

In an embodiment, the MenC saccharide, where present, has a molecularweight of 100-200 kDa, 50-100 kDa, 100-150 kDa, 101-130 kDa, 150-210 kDaor 180-210 kDa as determined by MALLS.

In an embodiment the MenY saccharide, where present, has a molecularweight of 60-190 kDa, 70-180 kDa, 80-170 kDa, 90-160 kDa, 100-150 kDa or110-140 kDa, 50-100 kDa, 100-140 kDa, 140-170 kDa or 150-160 kDa asdetermined by MALLS.

In an embodiment the MenW saccharide, where present, has a molecularweight of 60-190 kDa, 70-180 kDa, 80-170 kDa, 90-160 kDa, 100-150 kDa,110-140 kDa, 50-100 kDa or 120-140 kDa as determined by MALLS.

The molecular weights of the saccharide refers to the molecular weightof the polysaccharide measured prior to conjugation and is measured byMALLS.

In an embodiment the N. meningitidis saccharides are nativepolysaccharides or native polysaccharides which have reduced in sizeduring a normal extraction process.

In an embodiment, the N. meningitidis saccharides are sized bymechanical cleavage, for instance by microfluidisation or sonication.Microfluidisation and sonication have the advantage of decreasing thesize of the larger native polysaccharides sufficiently to provide afilterable conjugate.

In an embodiment, the polydispersity of the saccharide is 1-1.5, 1-1.3,1-1.2, 1-1.1 or 1-1.05 and after conjugation to a carrier protein, thepolydispersity of the conjugate is 1.0-2.5, 1.0-2.0, 1.0-1.5, 1.0-1.2,1.5-2.5, 1.7-2.2 or 1.5-2.0. All polydispersity measurements are byMALLS.

For MALLS analysis of meningococcal saccharides, two columns (TSKG6000and 5000PWxl TOSOH Bioscience) may be used in combination and thesaccharides are eluted in water. Saccharides are detected using a lightscattering detector (for instance Wyatt Dawn DSP equipped with a 10 mWargon laser at 488 nm) and an inferometric refractometer (for instanceWyatt Otilab DSP equipped with a P100 cell and a red filter at 498 nm).

In an embodiment, the MenA saccharide, where present is at leastpartially O-acetylated such that at least 50%, 60%, 70%, 80%, 90%, 95%or 98% of the repeat units are O-acetylated at least one position.O-acetylation is for example present at least at the O-3 position.

In an embodiment, the MenC saccharide, where present is at leastpartially O-acetylated such that at least 30%. 40%, 50%, 60%, 70%, 80%,90%, 95% or 98% of (α2→9)-linked NeuNAc repeat units are O-acetylated atleast one or two positions. O-acetylation is for example present at theO-7 and/or O-8 position.

In an embodiment, the MenW saccharide, where present is at leastpartially O-acetylated such that at least 30%. 40%, 50%, 60%, 70%, 80%,90%, 95% or 98% of the repeat units are O-acetylated at least one or twopositions. O-acetylation is for example present at the O-7 and/or O-9position.

In an embodiment, the MenY saccharide, where present is at leastpartially O-acetylated such that at least 40%, 50%, 60%, 70%, 80%, 90%,95% or 98% of the repeat units are O-acetylated at least one or twopositions. O-acetylation is present at the 7 and/or 9 position.

The percentage of O-acetylation refers to the percentage of the repeatunits containing O-acetylation. This may be measured in the saccharideprior to conjugate and/or after conjugation.

A further aspect of the invention is a vaccine comprising theimmunogenic composition of the invention and a pharmaceuticallyacceptable excipient.

Optionally, the immunogenic composition or vaccine contains an amount ofan adjuvant sufficient to enhance the immune response to the immunogen.Suitable adjuvants include, but are not limited to, aluminium salts(aluminium phosphate or aluminium hydroxide), squalene mixtures (SAF-1),muramyl peptide, saponin derivatives, mycobacterium cell wallpreparations, monophosphoryl lipid A, mycolic acid derivatives,non-ionic block copolymer surfactants, Quil A, cholera toxin B subunit,polphosphazene and derivatives, and immunostimulating complexes (ISCOMs)such as those described by Takahashi et al. (1990) Nature 344:873-875.

For the HibMen combinations discussed above, it may be advantageous forthe Hib and Men saccharide conjugates not to be adsorbed to aluminiumsalt adjuvant or any adjuvant at all.

In an embodiment, the immunogenic composition is unadjuvanted. For thepurpose of the invention, “unadjuvanted” means that an adjuvantcomponent which is not an antigenic component in its own right is notpresent in the immunogenic composition.

In an embodiment of the invention HepB is adsorbed to aluminiumphosphate (WO 93/24148).

In an embodiment, the immunogenic composition comprises a Hib saccharideconjugated to tetanus toxoid via a linker and MenC saccharide conjugatedto tetanus toxoid either directly or through a linker and MenAsaccharide conjugated to tetanus toxoid either directly or through alinker.

In an embodiment, the immunogenic composition of the invention isbuffered at, or adjusted to, between pH 7.0 and 8.0, pH 7.2 and 7.6 oraround or exactly pH 7.4.

The immunogenic composition or vaccines of the invention are optionallylyophilised in the presence of a stabilising agent for example a polyolsuch as sucrose or trehalose.

As with all immunogenic compositions or vaccines, the immunologicallyeffective amounts of the immunogens must be determined empirically.Factors to be considered include the immunogenicity, whether or not theimmunogen will be complexed with or covalently attached to an adjuvantor carrier protein or other carrier, route of administrations and thenumber of immunizing dosages to be administered. Such factors are knownin the vaccine art and it is well within the skill of immunologists tomake such determinations without undue experimentation.

The active agent can be present in varying concentrations in thepharmaceutical composition or vaccine of the invention. Typically, theminimum concentration of the substance is an amount necessary to achieveits intended use, while the maximum concentration is the maximum amountthat will remain in solution or homogeneously suspended within theinitial mixture. For instance, the minimum amount of a therapeutic agentis optionally one which will provide a single therapeutically effectivedosage. For bioactive substances, the minimum concentration is an amountnecessary for bioactivity upon reconstitution and the maximumconcentration is at the point at which a homogeneous suspension cannotbe maintained. In the case of single-dosed units, the amount is that ofa single therapeutic application. Generally, it is expected that eachdose will comprise 1-100 μg of protein antigen, optionally 5-50 μg or5-25 μg. The vaccine preparations of the present invention may be usedto protect or treat a mammal (for example a human patient) susceptibleto infection, by means of administering said vaccine via systemic ormucosal route. A human patient is optionally an infant (under 12months), a toddler (12-24, 12-16 or 12-14 months), a child (2-10, 3-8 or3-5 years) an adolescent (12-25, 14-21 or 15-19 years) or an adult (anyage over 12, 15, 18 or 21). These administrations may include injectionvia the intramuscular, intraperitoneal, intradermal or subcutaneousroutes; or via mucosal administration to the oral/alimentary,respiratory, genitourinary tracts. Intranasal administration of vaccinesfor the treatment of pneumonia or otitis media is preferred (asnasopharyngeal carriage of pneumococci can be more effectivelyprevented, thus attenuating infection at its earliest stage). Althoughthe vaccine of the invention may be administered as a single dose,components thereof may also be co-administered together at the same timeor at different times (for instance if saccharides are present in avaccine these could be administered separately at the same time or 1-2weeks after the administration of a bacterial protein vaccine foroptimal coordination of the immune responses with respect to eachother). In addition to a single route of administration, 2 differentroutes of administration may be used. For example, viral antigens may beadministered ID (intradermal), whilst bacterial proteins may beadministered IM (intramuscular) or IN (intranasal). If saccharides arepresent, they may be administered IM (or ID) and bacterial proteins maybe administered IN (or ID). In addition, the vaccines of the inventionmay be administered IM for priming doses and IN for booster doses.

Vaccine preparation is generally described in Vaccine Design (“Thesubunit and adjuvant approach” (eds Powell M. F. & Newman M. J.) (1995)Plenum Press New York). Encapsulation within liposomes is described byFullerton, U.S. Pat. No. 4,235,877.

A further aspect of the invention is a vaccine kit for concomitant orsequential administration comprising two multi-valent immunogeniccompositions for conferring protection in a host against disease causedby Bordetella pertussis, Clostridium tetani, Corynebacteriumdiphtheriae, Haemophilus influenzae and Neisseria meningitidis. Forexample, the kit optionally comprises a first container comprising oneor more of:

tetanus toxoid (TT),

diphtheria toxoid (DT), and

whole cell pertussis components and

optionally further comprising Hepatitis B surface antigen

and a second container comprising:

Hib saccharide conjugate, and

at least one, two, three or four N. meningitidis saccharideconjugate(s),

wherein the saccharide dose of the Hib conjugate is less than 5 μg or 4μg, or 1-4 μg or 1-3 μg, or 2-4 μg or 2-3 μg or around or exactly 2.5 μgand optionally the saccharide dose of the or each of the at least orexactly one, two, three or four additional bacterial saccharideconjugate(s) (for example N. meningitidis saccharide conjugate(s)) isless than 10 μg, 9 μg, 8 μg, 7 μg, 6 μg, 5 μg or 4 μg, or between 1-10μg, 1-8 μg, 1-6 μg, 1-5 μg, 1-4 μg, 1-3 μg, or 2-4 μg or 2-3 μg or aboutor exactly 2.5 μg.

Examples of the Hib conjugate and the at least one additional bacterialsaccharide (for example N. meningitidis saccharide) conjugate(s) are asdescribed above. Any of the properties of the conjugates described abovecan be present in a vaccine kit.

Optionally, the vaccine kits of the invention comprise a thirdcomponent. For example, the kit optionally comprises a first containercomprising one or more of:

tetanus toxoid (TT),

diphtheria toxoid (DT), and

whole cell pertussis components and

optionally comprises Hepatitis B surface antigen;

and a second container comprising:

one or more conjugates of a carrier protein and a capsular saccharidefrom Streptococcus pneumoniae [where the capsular saccharide isoptionally from a pneumococcal serotype selected from the groupconsisting of 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14,15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F].

and a third container comprising:

Hib saccharide conjugate, and

at least one N. meningitidis saccharide conjugate(s),

wherein the saccharide dose of the Hib conjugate is less than 5 μg or 4μg, or 1-4 μg or 1-3 μg, or 2-4 μg or 2-3 μg or around or exactly 2.5 μgand optionally the saccharide dose of the or each of the at least orexactly one, two, three or four additional bacterial saccharideconjugate(s) (for example N. meningitidis saccharide conjugate(s)) isless than 10 μg, 9 μg, 8 μg, 7 μg, 6 μg, 5 μg or 4 μg, or between 1-10μg, 1-8 μg, 1-6 μg, 1-5 μg, 1-4 μg, 1-3 μg, or 2-4 μg or 2-3 μg or aboutor exactly 2.5 μg.

Immunogenic compositions comprising meningococcal conjugates, forexample HibMenC, HibMenAC, HibMenAW, HibMenAY, HibMenCW, HibMenCY,HibMenWY, MenAC, MenAW, MenAY, MenCW, MenCY, MenWY or MenACWY, includingkits of similar composition to those described above, optionallycomprise antigens from measles and/or mumps and/or rubella and/orvaricella. For example, the meningococcal immunogenic compositioncontains antigens from measles, mumps and rubella or measles, mumps,rubella and varicella. In an embodiment, these viral antigens areoptionally present in the same container as the meningococcal and/or Hibsaccharide conjugate(s). In an embodiment, these viral antigens arelyophilised.

A further aspect of the invention is a process for making theimmunogenic composition of the invention, comprising the step of mixinga Hib saccharide conjugate with at least one additional bacterial (forexample N. meningitidis) saccharide conjugate(s) and a further antigenselected from the group consisting of whole cell pertussis and hepatitisB surface antigen to form a composition wherein the saccharide dose ofthe Hib conjugate is less than 5 μg or 4 μg, or between 1-4 μg or 1-3μg, or 2-4 μg or 2-3 μg or around or exactly 2.5 μg and optionally thesaccharide dose of the or each of the at least or exactly one, two,three or four additional bacterial (for example N. meningitidis)saccharide conjugate(s) is less than 10 μg, 8 μg, 7 μg, 6 μg, 5 μg or 4μg, or between 1-10 μg, 1-8 μg, 1-6 μg, 1-5 μg, 1-4 μg, 1-3 μg, or 2-4μg or 2-3 μg or about or exactly 2.5 μg.

A further aspect of the invention is a method of immunising a human hostagainst disease caused by Haemophilus influenzae and optionally N.meningitidis infection comprising administering to the host animmunoprotective dose of the immunogenic composition or vaccine or kitof the invention.

A further aspect of the invention is an immunogenic composition of theinvention for use in the treatment or prevention of disease caused byHaemophilus influenzae and/or N. meningitidis.

A further aspect of the invention is use of the immunogenic compositionor vaccine or kit of the invention in the manufacture of a medicamentfor the treatment or prevention of diseases caused by Haemophilusinfluenzae and/or N. meningitidis.

The terms “comprising”, “comprise” and “comprises” herein are intendedby the inventors to be optionally substitutable with the terms“consisting of”, “consist of” and “consists of”, respectively, in everyinstance.

All references or patent applications cited within this patentspecification are incorporated by reference herein.

The invention is illustrated in the accompanying examples. The examplesbelow are carried out using standard techniques, which are well knownand routine to those of skill in the art, except where otherwisedescribed in detail. The examples are illustrative, but do not limit theinvention.

EXAMPLES Example 1 Preparation of Polysaccharide Conjugates

The covalent binding of Haemophilus influenzae (Hib) PRP polysaccharideto TT was carried out by a coupling chemistry developed by Chu et al(Infection and Immunity 1983, 40 (1); 245-256). Hib PRP polysaccharidewas activated by adding CNBr and incubating at pH10.5 for 6 minutes. ThepH was lowered to pH8.75 and adipic acid dihyrazide (ADH) was added andincubation continued for a further 90 minutes. The activated PRP wascoupled to purified tetanus toxoid via carbodiimide condensation using1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide (EDAC). EDAC was added tothe activated PRP to reach a final ratio of 0.6 mg EDAC/mg activatedPRP. The pH was adjusted to 5.0 and purified tetanus toxoid was added toreach 2 mg TT/mg activated PRP. The resulting solution was left forthree days with mild stirring. After filtration through a 0.45 μmmembrane, the conjugate was purified on a sephacryl S500HR (Pharmacia,Sweden) column equilibrated in 0.2M NaCl.

MenC-TT conjugates were produced using native polysaccharides (of over150 kDa as measured by MALLS) or were slightly microfluidised. MenA-TTconjugates were produced using either native polysaccharide or slightlymicrofluidised polysaccharide of over 60 kDa as measured by the MALLSmethod of example 2. MenW and MenY-TT conjugates were produced usingsized polysaccharides of around 100-200 kDa as measured by MALLS (seeexample 2). Sizing was by microfluidisation using a homogenizerEmulsiflex C-50 apparatus. The polysaccharides were then filteredthrough a 0.2 μm filter.

Activation and coupling were performed as described in WO096/29094 andWO 00/56360. Briefly, the polysaccharide at a concentration of 10-20mg/ml in 2M NaCl pH 5.5-6.0 was mixed with CDAP solution (100 mg/mlfreshly prepared in acetonitrile/WFI, 50/50) to a finalCDAP/polysaccharide ratio of 0.75/1 or 1.5/1. After 1.5 minutes, the pHwas raised with sodium hydroxide to pH10.0. After three minutes tetanustoxoid was added to reach a protein/polysaccharide ratio of 1.5/1 forMenW, 1.2/1 for MenY, 1.5/1 for MenA or 1.5/1 for MenC. The reactioncontinued for one to two hours.

After the coupling step, glycine was added to a final ratio ofglycine/PS (w/w) of 7.5/1 and the pH was adjusted to pH9.0. The mixturewas left for 30 minutes. The conjugate was clarified using a 10 μmKleenpak filter and was then loaded onto a Sephacryl S400HR column usingan elution buffer of 150 mM NaCl, 10 mM or 5 mM Tris pH7.5. Clinicallots were filtered on an Opticap 4 sterilizing membrane. The resultantconjugates had an average polysaccharide:protein ratio of 1:1-1:5 (w/w).

In order to conjugate MenA capsular polysaccharide to tetanus toxoid viaa spacer, the following method was used. The covalent binding of thepolysaccharide and the spacer (ADH) is carried out by a couplingchemistry by which the polysaccharide is activated under controlledconditions by a cyanylating agent, 1-cyano-4-dimethylamino-pyridiniumtetrafluoroborate (CDAP). The spacer reacts with the cyanylated PSthrough its hydrazino groups, to form a stable isourea link between thespacer and the polysaccharide.

A 10 mg/ml solution of MenA was treated with a freshly prepared 100mg/ml solution of CDAP in acetonitrile/water (50/50 (v/v)) to obtain aCDAP/MenA ratio of 0.75 (w/w). After 1.5 minutes, the pH was raised topH 10.0. Three minutes later, ADH was added to obtain an ADH/MenA ratioof 8.9. The pH of the solution was decreased to 8.75 and the reactionproceeded for 2 hours.

Prior to the conjugation reaction, the purified TT solution and thePSA_(AH) solution were diluted to reach a concentration of 10 mg/ml forPSA_(AH) and 10 mg/ml for TT.

EDAC was added to the PS_(AH) solution in order to reach a final ratioof 0.9 mg EDAC/mg PSA_(AH). The pH was adjusted to 5.0. The purifiedtetanus toxoid was added with a peristaltic pump (in 60 minutes) toreach 2 mg TT/mg PSA_(AH). The resulting solution was left 60 min at+25° C. under stirring to obtain a final coupling time of 120 min. Theconjugate was clarified using a 10 μm filter and was purified using aSephacryl S400HR column.

Example 2 Determination of Molecular Weight Using MALLS

Detectors were coupled to a HPLC size exclusion column from which thesamples were eluted. On one hand, the laser light scattering detectormeasured the light intensities scattered at 16 angles by themacromolecular solution and on the other hand, an interferometricrefractometer placed on-line allowed the determination of the quantityof sample eluted. From these intensities, the size and shape of themacromolecules in solution can be determined.

The mean molecular weight in weight (M_(w)) is defined as the sum of theweights of all the species multiplied by their respective molecularweight and divided by the sum of weights of all the species.

-   -   a) Weight-average molecular weight: -Mw-

$M_{w} = {\frac{\sum{W_{i} \cdot M_{i}}}{\sum W_{i}} = \frac{m_{2}}{m_{1}}}$

-   -   b) Number-average molecular weight: -Mn-

$M_{n} = {\frac{\sum{N_{i} \cdot M_{i}}}{\sum N_{i}} = \frac{m_{1}}{m_{0}}}$

-   -   c) Root mean square radius: -Rw- and R²w is the square radius        defined by:

R²w${{{or}\left( r^{2} \right)}w} = \frac{\sum{m_{i} \cdot r_{i}^{2}}}{\sum m_{i}}$

-   -   -   (-m_(i)- is the mass of a scattering centre i and -r_(i)- is            the distance between the        -   scattering centre i and the center of gravity of the            macromolecule).

    -   d) The polydispersity is defined as the ratio -Mw/Mn-.

Meningococcal polysaccharides were analysed by MALLS by loading onto twoHPLC columns (TSKG6000 and 5000PWxl TOSOH Bioscience) used incombination. 250 of the polysaccharide were loaded onto the column andwas eluted with 0.75 ml of filtered water. The polysaccharides aredetected using a light scattering detector (Wyatt Dawn DSP equipped witha 10 mW argon laser at 488 nm) and an inferometric refractometer WyattOtilab DSP equipped with a P100 cell and a red filter at 498 nm).

The molecular weight polydispersities and recoveries of all samples werecalculated by the Debye method using a polynomial fit order of 1 in theAstra 4.72 software.

Example 3 Phase II Clinical Trial on HibMenAC-TT Conjugate Vaccine Mixedwith DTPw-HepB

Study Design:

Open, randomized (1:1:1:1:1), single centre study with five groups. Thefive groups received the following vaccination regimen respectively, at6, 10 and 14 weeks of age.

-   -   Tritanrix™-HepB/Hib-MenAC 2.5/2.5/2.5: henceforth referred to as        2.5/2.5/2.5    -   Tritanrix™-HepB/Hib-MenAC 2.5/5/5: henceforth referred to as        2.5/5/5    -   Tritanrix™-HepB/Hib-MenAC 5/5/5: henceforth referred to as 5/5/5    -   Tritanrix™-HepB+Hiberix™: henceforth referred to as Hiberix    -   Tritanrix™-HepB/Hiberix™+Meningitec™: henceforth referred to as        Meningitec

Blood samples were taken at the time of the first vaccine dose (Pre) andone month after the third vaccine dose

(Post-dose 3).

Tritanrix™ is a DTPw vaccine marketted by GlaxoSmithKline BiologicalsS.A.

105 subjects were used in each of the five groups giving a total of 525subjects in the study.

TABLE 1 Components per dose (0.5 ml) 2.5/2.5/2.5* 2.5/5/5 5/5/5 Hibcapsular polysaccharide PRP 2.5 μg 2.5 μg   5 μg conjugated to tetanustoxoid (TT) Neisseria meningitidis A capsular 2.5 μg 5 μg 5 μgpolysaccharide (PSA) conjugated to TT Neisseria meningitidis C capsular2.5 μg 5 μg 5 μg polysaccharide (PSC) conjugated to TT *The 2.5/2.5/2.5vaccine was a dose dilution of GSK Biologicals' Hib-MenAC 5/5/5 vaccinecontaining 2.5 μg of each of PRP-TT, MenA-TT and MenC-TT.

The Hib-MenAC vaccine formulations were mixed extemporaneously withTritanirix-HepB. GSK Biologicals' combined diphtheria-tetanus-whole cellBordetella pertussis—hepatitis B (DTPw-HB) vaccine (Tritanrix-HepB)contains not less than 30 International Units (IU) of diphtheria toxoid,not less than 60 IU of tetanus toxoid, not less than 41U of killedBordetella pertussis and 10 μg of recombinant hepatitis B surfaceantigen.

Reference Therapy, Dose, Mode of Administration, Lot No.:

Vaccination Schedule/Site:

One group received Tritanrix.-HepB vaccine intramuscularly in the leftthigh and Hiberix™ intramuscularly in the right thigh at 6, 10 and 14weeks of age. Another group received Tritanrix™-HepB/Hiberix™ vaccineintramuscularly in the left thigh and Meningitec™ vaccineintramuscularly in the right thigh at 6, 10 and 14 weeks of age.

Vaccine/Composition/Dose/Lot Number:

The Tritanrix™-HepB vaccine used was as described above.

One dose (0.5 ml) of GSK Biologicals' Haemophilus influenzae type bconjugate vaccine: Hiberix™ contained 10 μg of PRP conjugated to tetanustoxoid. In the Hiberix Group, it was mixed with sterile diluent and inthe Meningitec Group it was mixed with Tritanrix-HepB.

One dose (0.5 ml) of Wyeth Lederle's MENINGITEC™ vaccine contained: 10μg of capsular polysaccharide of meningococcal group C conjugated to 15μg of Corynebacterium diphtheria CRM197 protein and aluminium as salts.

Results—Immune Responses Generated Against Hib, MenA and MenC

TABLE 2a Anti-PRP (μg/ml) Group 2.5/2.5/2.5 2.5/5/5 5/5/5 Hiberix ™Meningitec ™ % 95% CL % 95% CL % 95% CL % 95% CL % 95% CL GMC/T LL ULGMC/T LL UL GMC/T LL UL GMC/T LL UL GMC/T LL UL % ≧0.15 100 96.5 10099.0 94.8 100 100 96.5 100 100 96.5 100 100 96.5 100 GMC 20.80 15.9627.10 22.62 17.72 28.88 19.36 15.33 24.46 38.55 29.93 49.64 10.94 8.6213.88

TABLE 2b SBA-MenC Group 2.5/2.5/2.5 2.5/5/5 5/5/5 Hiberix ™ Meningitec ™% 95% CL % 95% CL % 95% CL % 95% CL % 95% CL GMC/T LL UL GMC/T LL ULGMC/T LL UL GMC/T LL UL GMC/T LL UL % ≧1:8 99 94.7 100 100 96.5 100 10096.5 100 2.9 0.6 8.4 100 96.5 100 GMT 3132 2497 3930 4206 3409 5189 36973118 4384 4.7 3.9 5.6 4501 3904 5180

TABLE 2c SBA MenA Group 2.5/2.5/2.5 2.5/5/5 5/5/5 Hiberix ™ Meningitec ™% 95% CL % 95% CL % 95% CL % 95% CL % 95% CL GMC/T LL UL GMC/T LL ULGMC/T LL UL GMC/T LL UL GMC/T LL UL % ≧1:8 99.7 91.9 99.7 100 95.8 100100 96.2 100 6.8 2.5 14.3 9.1 4.0 17.1 GMT 316.7 251.4 398.9 418.5 358.6488.5 363 310.5 424.4 5.6 4.3 7.4 5.6 4.4 7.2

TABLE 2d Anti-PSC (μg/ml) 2.5/2.5/2.5 2.5/5/5 5/5/5 Hiberix ™Meningitec ™ % 95% CL % 95% CL % 95% CL % 95% CL % 95% CL Group GMC/T LLUL GMC/T LL UL GMC/T LL UL GMC/T LL UL GMC/T LL UL % ≧0.3 100 96.5 100100 96.4 100 100 96.5 100 8.2 3.6 15.6 100 96.5 100 GMC 49.03 43.2455.59 71.11 62.49 80.92 61.62 54.88 69.20 0.17 0.15 0.19 58.02 51.4265.46

TABLE 2e Anti-PSA (μg/ml) 2.5/2.5/2.5 2.5/5/5 5/5/5 Hiberix ™Meningitec ™ % 95% CL % 95% CL % 95% CL % 95% CL % 95% CL Group GMC/T LLUL GMC/T LL UL GMC/T LL UL GMC/T LL UL GMC/T LL UL % ≧0.3 100 96.4 100100 96.5 100 99.0 94.8 100 1.0 0.0 5.4 5.9 2.2 12.5 GMC 18.10 15.3421.35 26.51 22.93 30.79 23.40 20.05 27.30 0.15 0.15 0.15 0.17 0.15 0.18

TABLE 2f Anti-BPT (EL.U/ml) 2.5/2.5/2.5 2.5/5/5 5/5/5 Hiberix ™Meningitec ™ % 95% CL % 95% CL % 95% CL % 95% CL % 95% CL Group GMC/T LLUL GMC/T LL UL GMC/T LL UL GMC/T LL UL GMC/T LL UL % VR 100 96.4 100 9893.1 99.8 94.2 87.8 97.8 100 96.3 100 99.0 94.7 100 GMC 108.2 94.4 123.981.4 70.5 94.0 60.1 51.0 70.8 110.2 96.1 126.3 86.7 75.4 99.7

TABLE 2g Anti-HBs (mIU/ml) 2.5/2.5/2.5 2.5/5/5 5/5/5 Hiberix ™Meningitec ™ % 95% CL % 95% CL % 95% CL % 95% CL % 95% CL Group GMC/T LLUL GMC/T LL UL GMC/T LL UL GMC/T LL UL GMC/T LL UL % ≧10 92.1 85.0 96.583.8 75.1 90.5 82.5   73.8 89.3 91.6 84.1 96.3 83.2 74.4 89.9 GMC 128.695.4 173.4 71.8 53.2 96.9 55.3.1 41.0 74.6 104.5 76.0 143.7 71.1 52.197.1

Conclusion

Tables 2f and 2g demonstrate that, when HibMenAC is mixed with DTPwHepBin a vaccine, a dose of 2.5/2.5/2.5 for Hib/MenA/MenC gives a higherimmune response against pertussis toxoid and Hepatitis surface antigenthan a dose of 2.5/5/5 which in turn generates a higher immune responseagainst pertussis toxoid and Hepatitis B surface antigen than a dose of5/5/5. Table 2a-e demonstrate that a low dose of HibMenA and MenC stillachieves a good immune response against Hib, MenA and MenC with 99-100%of patients achieving an immune response above the chosen thresholds.

Example 4 HibMenAC Clinical Trial—Priming with HibMenAC Conjugates

A phase II, open, randomized study was carried out to assess the immunememory induced by primary vaccination course of Tritanrix™-HepB/HibMenACvaccine, and to assess the immunogenicity and reactogenicity of abooster dose of GSK Biologicals' Tritanrix™-HepB vaccine mixed witheither GSK Biologicals' Hib-MenAC conjugate vaccine or GSK Biologicals'Hib₂₅ vaccine at 15 to 18 months of age in subjects primed withTritanrix™-HepB/Hib-MenAC. Five groups received the primary vaccinationregimens at 6, 10 and 14 weeks of age as presented in table 3.

TABLE 3 At 15 to 18 months of Primary vaccination Grp At 10 months ofage age Treatment groups Tritanrix ™-HepB/Hib-MenAC 1 ⅕^(th) dose ofMencevax ™ AC (10 μg MenA & 10 μg MenC) and 10 μgTritanrix ™-HepB/Hib_(2.5) 2.5/2.5/2.5 of Plain PRP 2 —Tritanrix ™-HepB/Hib_(2.5) Tritanrix ™-HepB/Hib- 3 ⅕^(th) dose ofMencevax ™ AC (10 μg MenA & 10 μg MenC) and 10 μgTritanrix ™-HepB/Hib_(2.5) MenAC 5/5/5 of Plain PRP 4 —Tritanrix ™-HepB/Hib_(2.5) Tritanrix ™-HepB/Hib-MenAC 5 ⅕^(th) dose ofMencevax ™ AC (10 μg MenA & 10 μg MenC) and 10 μgTritanrix ™-HepB/Hib_(2.5) 2.5/5/5 of Plain PRP 6 —Tritanrix ™-HepB/Hib_(2.5) Control groups Tritanrix ™-HepB + Hiberix ™ 7⅕^(th) dose of Mencevax ™ AC (10 μg MenA & 10 μg MenC) and 10 μgTritanrix ™-HepB/Hib- of Plain PRP MenAC 8 — Tritanrix ™-HepB/Hib- MenACTritanrix ™-HepB/Hiberix ™ + 9 ⅕^(th) dose of Mencevax ™ AC (10 μg MenA& 10 μg MenC) and 10 μg Tritanrix ™-HepB/Hib- Meningitec ™ of Plain PRPMenAC 10 — Tritanrix ™-HepB/Hib- MenAC

Blood samples were taken from Groups 1, 3, 5, 7 and 9 at the time of theplain polysaccharide (PS) booster (i.e. Pre-PS-Month 10) and one monthafter the plain polysaccharide booster (i.e. Post-PS-Month 11).

Note:

The immunogenicity results obtained in the five groups who received theplain polysaccharide booster (i.e. Groups 1, 3, 5, 7 and 9) have beenpresented.

Number of Subjects:

Planned: 450 (45 subjects per group)

Enrolled:

In Groups 1, 3, 5, 7 and 9 receiving the plain polysaccharide booster atotal of 193 subjects (42 in Group 1, 39 in Group 3, 37 in Group 5, 36in Group 7 and 39 in Group 9) were enrolled. Completed: Not applicable

Immunogenicity:

Total enrolled cohort=193 subjects

Note:

In this study the total enrolled cohort=total vaccinated cohort.

Diagnosis and Criteria for Inclusion:

A male or female subject aged 10 months of age who had completed thethree-dose primary vaccination course described in example 1, free ofobvious health problems, who had not received previous boostervaccination against diphtheria, tetanus, pertussis, hepatitis B,meningococcal serogroups A or C and/or Hib disease since the studyconclusion visit of the primary study. Written informed consent wasobtained from the parent/guardian of the subject prior to study entry.

Study Vaccines, Dose, Mode of Administration, Lot No.:

All vaccines used in this study were developed and manufactured by GSKBiologicals.

Vaccination Schedule/Site:

Subjects in Groups 1, 3, 5, 7 and 9 received the combined polysaccharideA and polysaccharide C vaccine, ⅕^(th) dose of Mencevax™ AC and 10 μg ofplain PRP as an intramuscular injection in the left and rightanterolateral thigh at 10 months of age, respectively.

Duration of Treatment:

The duration of the entire study was approximately 6 to 9 months persubject which included the booster vaccination administered at 15 to 18months of age. Interim analysis was done at Month 11 (i.e. one monthafter administration of the plain polysaccharide booster at Month 10).

Criteria for Evaluation:

Prior to and one month after administration of the plain polysaccharidebooster the criteria for evaluation for Groups 1, 3, 5, 7 and 9 were asfollows

-   -   SBA-MenA antibody titre≧1:8    -   SBA-MenC antibody titre≧1:8    -   Anti-PSA antibody concentration≧0.3 μg/ml    -   Anti-PSC antibody concentration≧0.3 μg/ml    -   Anti-PRP antibody concentration≧0.15 μg/ml.

Statistical Methods:

This interim analysis was based on the total enrolled cohort. Allanalyses were purely descriptive and no statistical inference on anyendpoints was calculated. Analyses were performed only for the fivegroups (i.e. Groups 1, 3, 5, 7 and 9) that received the plainpolysaccharide booster at 10 months of age. Though these five groupswere sub-groups of the main groups in the primary study, the results arepresented as per the primary study group allocation.

Analysis of Immunogenicity:

The results obtained at three time points have been presented in thisexample namely—one month after the third vaccine dose in the primaryvaccination study (Example 1), prior to the administration of thepolysaccharide booster (i.e. at 10 months of age) for evaluation of thepersistence of immune response after primary vaccination and one monthafter the administration of the polysaccharide booster (i.e. at 11months of age) for evaluation of immune memory induced by primaryvaccination. At each time point: Geometric Mean antibody Concentrationsor Titres (GMCs or GMTs) with 95% confidence intervals (CIs) weretabulated for serum bactericidal assay (SBA)-MenC, SBA-MenA, anti-PSC,anti-PSA and anti-PRP. Seropositivity or seroprotection rates with exact95% Cls were calculated for each antibody. Antibody concentrations ortitres prior to polysaccharide booster & one month post-polysaccharidebooster were investigated using reverse cumulative curves (RCCs) foreach antigen and serotype.

Results

Demography Results:

The mean age of the total enrolled cohort was 43.2 weeks with a standarddeviation of 6.5 weeks. The male to female ratio was 1.3 (110/83). Allsubjects belonged to either the East Asian or South-East Asian race.

Immunogenicity Results:

The immunogenicity results for the total enrolled cohort are presentedin the table 4.

TABLE 4 95% CI 95% CI Antibody Group Timing (LL, UL) GMC/GMT (LL, UL) aAnti-PRP 2.5/2.5/2.5 PIII(M3) 100.0 91.6 100.0 17.872 11.358 28.123 (%≧0.15 μg/ml) PRE-PS 97.5 86.8 99.9 6.940 4.402 10.941 POST-PS 100.0 91.6100.0 66.510 38.690 114.334 5/5/5 PIII(M3) 100.0 91.0 100.0 17.30611.477 26.095 PRE-PS 94.9 82.7 99.4 4.520 2.946 6.937 POST-PS 100.0 91.0100.0 44.418 26.595 74.186 2.5/5/5 PIII(M3) 100.0 90.5 100.0 22.48415.217 33.223 PRE-PS 100.0 89.7 100.0 5.092 3.290 7.883 POST-PS 100.090.5 100.0 54.244 32.251 91.234 Hiberix ™ PIII(M3) 100.0 90.3 100.030.106 18.316 49.485 PRE-PS 100.0 90.3 100.0 5.105 3.238 8.049 POST-PS100.0 90.3 100.0 37.049 21.335 64.336 Meningitec ™ PIII(M3) 100.0 91.0100.0 12.257 8.234 18.246 PRE-PS 100.0 91.0 100.0 4.227 2.804 6.372POST-PS 100.0 91.0 100.0 24.354 15.308 38.747 SBA-MenA (% ≧1:8)2.5/2.5/2.5 PIII(M3) 97.1 84.7 99.9 342.3 230.7 507.9 PRE-PS 91.7 77.598.2 161.9 93.9 279.1 POST-PS 100.0 88.4 100.0 737.2 577.3 941.4 5/5/5PIII(M3) 100.0 90.0 100.0 394.6 297.8 523.0 PRE-PS 94.3 80.8 99.3 193.2126.7 294.7 POST-PS 96.7 82.8 99.9 720.8 479.8 1082.7 2.5/5/5 PIII(M3)100.0 90.0 100.0 385.8 285.9 520.5 PRE-PS 88.2 72.5 96.7 162.7 95.8276.2 POST-PS 100.0 88.4 100.0 929.9 718.4 1203.6 Hiberix ™ PIII(M3)10.0 2.1 26.5 6.6 3.7 11.7 PRE-PS 72.7 54.5 86.7 96.9 46.0 204.1 POST-PS100.0 89.4 100.0 631.8 475.5 839.4 Meningitec ™ PIII(M3) 6.9 0.8 22.84.8 3.6 6.4 PRE-PS 80.0 63.1 91.6 119.7 62.7 228.3 POST-PS 92.1 78.698.3 449.9 271.7 745.0 SBA-MenC (% ≧1:8) 2.5/2.5/2.5 PIII(M3) 100.0 91.6100.0 3342.3 2466.9 4528.3 PRE-PS 90.5 77.4 97.3 322.3 190.2 546.1POST-PS 100.0 91.6 100.0 2713.5 1909.4 3856.2 5/5/5 PIII(M3) 100.0 91.0100.0 3863.1 3025.9 4932.1 PRE-PS 97.3 85.8 99.9 463.9 292.9 734.7POST-PS 100.0 91.0 100.0 2377.3 1665.4 3393.4 2.5/5/5 PIII(M3) 100.090.5 100.0 5339.0 3829.4 7443.6 PRE-PS 94.6 81.8 99.3 451.4 281.7 723.5POST-PS 100.0 90.3 100.0 2824.7 2048.1 3895.8 Hiberix ™ PIII(M3) 2.8 0.114.5 4.5 3.6 5.7 PRE-PS 5.7 0.7 19.2 4.8 3.6 6.4 POST-PS 17.6 6.8 34.59.8 4.8 19.7 Meningitec ™ PIII(M3) 100.0 91.0 100.0 4557.8 3539.3 5869.5PRE-PS 97.4 86.5 99.9 347.7 221.6 545.4 POST-PS 100.0 91.0 100.0 1557.71090.8 2224.4 b Anti-PSA 2.5/2.5/2.5 PIII(M3) 100.0 91.2 100.0 17.6413.52 23.02 (% ≧0.3 μg/ml) PRE-PS 92.5 79.6 98.4 1.79 1.22 2.62 POST-PS100.0 91.6 100.0 23.58 16.76 33.17 5/5/5 PIII(M3) 100.0 91.0 100.0 26.0620.30 33.45 PRE-PS 97.4 86.5 99.9 2.25 1.60 3.18 POST-PS 100.0 91.0100.0 24.13 17.64 33.01 2.5/5/5 PIII(M3) 100.0 90.3 100.0 24.03 18.8430.65 PRE-PS 91.2 76.3 98.1 1.47 0.99 2.19 POST-PS 100.0 90.5 100.022.68 15.81 32.54 Hiberix ™ PIII(M3) 0.0 0.0 10.3 0.15 0.15 0.15 PRE-PS5.6 0.7 18.7 0.16 0.15 0.17 POST-PS 75.8 57.7 88.9 1.03 0.55 1.93Meningitec ™ PIII(M3) 2.6 0.1 13.8 0.16 0.14 0.17 PRE-PS 7.7 1.6 20.90.16 0.15 0.18 POST-PS 66.7 49.8 80.9 0.84 0.49 1.42 Anti-PSC2.5/2.5/2.5 PIII(M3) 100.0 91.6 100.0 48.45 39.65 59.20 (% ≧0.3 μg/ml)PRE-PS 100.0 91.2 100.0 7.11 5.69 8.89 POST-PS 100.0 91.2 100.0 21.5517.24 26.94 5/5/5 PIII(M3) 100.0 91.0 100.0 56.42 48.16 66.11 PRE-PS100.0 91.0 100.0 8.32 6.74 10.28 POST-PS 100.0 90.0 100.0 22.32 18.2127.36 2.5/5/5 PIII(M3) 100.0 90.3 100.0 76.98 62.69 94.53 PRE-PS 100.089.7 100.0 8.64 6.93 10.77 POST-PS 100.0 90.5 100.0 24.75 19.37 31.61Hiberix ™ PIII(M3) 6.1 0.7 20.2 0.16 0.15 0.18 PRE-PS 0.0 0.0 9.7 0.150.15 0.15 POST-PS 100.0 90.3 100.0 8.05 5.73 11.30 Meningitec ™ PIII(M3)100.0 91.0 100.0 59.05 48.16 72.41 PRE-PS 100.0 91.0 100.0 7.33 5.519.75 POST-PS 100.0 90.7 100.0 17.13 13.38 21.94 95% CI: 95% confidenceinterval; LL: Lower Limit; UL: Upper Limit; GMC/GMT: Geometric meanconcentration/Geometric mean titre PIII(M 3): Post-vaccination bloodsample obtained one month after the third dose of the three-dose primaryvaccination PRE-PS: Blood sample obtained prior to plain polysaccharidebooster at Month 10 POST-PS: Blood sample obtained one month after theplain polysaccharide booster

Conclusion

The HibMenAC 2.5/5/5 conjugate vaccine formulation containing a loweramount of Hib tended to give a better immune memory response to MenA andMenC in SBA assays than the vaccine formulations containing equalamounts of all three conjugates. This can be seen from a comparison ofthe POST-PS readings. Therefore the use of the 2.5/5/5 formulation inpriming results in a superior immune memory response.

Looking at the PIII(M3) data, higher readings were seen for the 2.5/5/5formulation for Hib (22.5 v 17) and MenC (76v 48 or 56 and 5339 v 3342or 3863 by SBA).

Example 5a Clinical Trial using HibMenCY given Concomitantly withInfanrix Penta and Prevenar in Infants at 2, 4 and 6 Months

Study Design:

Phase II, open (partially double-blind*), randomized (1:1:1:1:1),controlled, multicentric study with five parallel groups who receivedconcomitant vaccines as follows as a 3-dose primary vaccination courseat age 2, 4 and 6 months:

-   -   Group Hib-MenCY 2.5/5/5: Hib-MenCY (2.5/5/5)+Infanrix®        penta+Prevenar®    -   Group Hib-MenCY 5/10/10: Hib-MenCY (5/10/10)+Infanrix®        penta+Prevenar®    -   Group Hib-MenCY 5/5/5: Hib-MenCY (5/5/5)+Infanrix®        penta+Prevenar®    -   Group Menjugate: Menjugate®+Act HIB®+Infanrix® penta**    -   Group ActHIB: ActHIB®+Infanrix® penta+Prevenar®

*Hib-MenCY (2.5/5/5) and Hib-MenCY (5/10/10) were administered in adouble-blind manner. The Hib-MenCY (5/5/5) formulation could not beadministered in a double blind as it was prepared by reconstituting aHib-MenCY (10/10/10) formulation with 1.0 ml diluent (half the solutionwas discarded and the remaining 0.5 ml was administered), whereas theHib-MenCY (2.5/5/5) and Hib-MenCY (5/10/10) formulations wereadministered after reconstitution with 0.5 ml diluent.

**Subjects from this group will be offered two doses of a licensedpneumococcal conjugate vaccine at the end of the booster study792014/002 according to prescribing information.

Blood samples (4.0 ml) were obtained from all subjects prior to and onemonth after completion of the primary vaccination course (Study Month 0and Study Month 5).

The study was planned to be on 400 subjects with 80 subjects in each ofthe five groups.

In study was completed with a total of 398 subjects (Group Hib-MenCY2.5/5/5: 80 Group Hib-MenCY 5/10/10: 81; Group Hib-MenCY 5/5/5: 78;Group Menjugate: 81; Group ActHIB: 78)

Vaccination Schedule/Site:

Three doses injected intramuscularly at two month intervals, atapproximately 2, 4 and 6 months of age as follows:

TABLE 5 Vaccines administered and site Group Vaccines administered leftthigh Vaccines administered right thigh Hib-MenCY 2.5/5/5 Hib-TT (2.5μg)-MenC-TT (5 μg)- DTPa-HBV-IPV (Infanrix ® MenY-TT penta): upper (5μg) Pneumococcal (Prevenar ®): lower Hib-MenCY 5/10/10 Hib-TT (5μg)-MenC-TT (10 μg)- DTPa-HBV-IPV (Infanrix ® MenY-TT penta): upper (10μg) Pneumococcal (Prevenar ®): lower Hib-MenCY 5/5/5 Hib-TT (5μg)-MenC-TT (5 μg)- DTPa-HBV-IPV (Infanrix ® MenY-TT penta): upper (5μg) Pneumococcal (Prevenar ®): lower Menjugate ® ActHIB ® DTPa-HBV-IPV(Infanrix ® penta): upper MenC (Menjugate ®): lower ActHIB ® ActHIB ®DTPa-HBV-IPV (Infanrix ® penta): upper Pneumococcal (Prevenar ®): lower

TABLE 6 Candidate vaccine formulation and lot numbers VaccineFormulation: contents/dose Presentation Lot no. (diluent lot no.)Hib-MenCY 2.5/5/5 H. influenzae type b capsular Lyophilized pellet inDCYH003A48 polysaccharide polyribosyl ribitol monodose vial (0.5 ml(01B20/22A) (PRP) 2.5 μg conjugated to after reconstitution tetanustoxoid (TT); with saline diluent) N. meningitidis serogroup C capsularpolysaccharide (PSC) 5 μg conjugated to TT; N. meningitidis serogroup Ycapsular polysaccharide (PSY) 5 μg conjugated to TT Hib-MenCY 5/10/10PRP 5 μg conjugated to TT; Lyophilized pellet in DCYH002A48 PSC 10 μgconjugated to TT; monodose vial (0.5 ml (01B20/22A) PSY 10 μg conjugatedto TT after reconstitution with saline diluent) Hib-MenCY 5/5/5 PRP 5 μgconjugated to TT; Lyophilized pellet in DCYH001A48 PSC 5 μg conjugatedto TT; monodose vial.* (01B20/22A) PSY 5 μg conjugated to TT *TheHib-MenCY 5/5/5 was prepared by dissolving Hib-MenCY 10/10/10formulation with 1.0 ml diluent; 0.5 ml was administered and theremaining 0.5 ml was discarded.

Criteria for Evaluation:

Immunogenicity: Measurement of titers/concentrations of antibodiesagainst each vaccine antigen prior to the first dose (Month 0) andapproximately one month after the third dose (Month 5) in all subjects.Determination of bactericidal antibody titers against N. meningitidisserogroups C and Y (SBA-MenC and SBA-MenY) by a bactericidal test (assaycut-offs: a dilution of 1:8 and 1:128) and ELISA measurement ofantibodies against N. meningitidis serogroups C and Y (anti-PSC andanti-PSY, assay cut-offs ≧0.3 μg/ml and ≧2 μg/ml), the Hibpolysaccharide PRP (anti-PRP, assay cut-offs ≧0.15 μg/ml and ≧1.0μg/ml), the three pertussis antigens (anti-PT, anti-FHA, anti-PRN, assaycut-off ≧5 EL.U/ml), antibodies to hepatitis B surface antigen(anti-HBs, assay cut-off ≧10 mlU/mL), diphtheria and tetanus toxoids(anti-diphtheria and anti-tetanus, assay cut-off 0.1 IU/ml);anti-poliovirus types 1, 2 and 3 (assay cut-off 1:8); seven pneumococcalserotypes anti-4, anti-6B, anti-9V, anti-14, anti-18C, anti-19F,anti-23F (assay cut-off 0.05 μg/ml). Primary vaccine response to thepertussis antigens was defined as seropositivity (detectable antibodies)after the third dose in subjects with previously undetectable antibodiesor at least maintenance of pre vaccination antibody concentration insubjects who were initially seropositive.

Safety (Criteria for evaluation): 8-day (Days 0 to 7) follow-up, afteradministration of each vaccine dose, of solicited local (pain, redness,swelling) and general (drowsiness, fever, irritability, and loss ofappetite) symptoms reported on diary cards by the parent(s)/guardian(s)of the subjects; 31 day (Days 0 to 30) follow-up, after each vaccinedose, of unsolicited non-serious adverse events; and of serious adverseevents (SAEs) during the entire study period.

Statistical Methods:

Immunogenicity

Geometric Mean antibody Concentrations or Titers (GMC/Ts) with 95%confidence intervals (CIs) were tabulated for each antigen. Calculationof GMC/Ts was performed by taking the anti-logarithm in base 10(anti-log 10) of the mean of the log 10 concentration or titertransformations. Antibody concentrations or titers below the assaycut-off were given an arbitrary value of half the cut-off for thepurpose of GMC/T calculation. Percentages of subjects with antibodyconcentration/titer above the specified assay cut-offs or with a vaccineresponse with exact 95% CI were calculated. Antibodyconcentrations/titers were investigated using reverse cumulativeantibody curves for each antigen post-vaccination. The distribution ofantibody concentration for the 7 pneumococcal antigens was tabulated.

The differences between the Hib-MenCY groups, compared with the controlgroup were evaluated in an exploratory manner for each antibody, exceptfor SBA-MenY and anti-PSY, in terms of (1) the difference between thecontrol group (minus) the Hib-MenCY groups for the percentage ofsubjects above the specified cut-offs or with a vaccine response withtheir standardized asymptotic 95% Cl, (2) the GMC or GMT ratios of thecontrol group over the Hib-MenCY groups with their 95% Cl. The controlgroup was Menjugate for SBA-MenC and anti-PSC; the control group for allother antigens was Group ActHIB. The same comparisons were done toevaluate the difference between each pair of Hib-MenCY formulations foranti-PRP, SBA-MenC, anti-PSC, SBA-MenY, anti-PSY and anti-tetanusantibodies.

Seroprotection/Seropositivity Rates &GMC/Ts (ATP Cohort forImmunogenicity)

TABLE 7a Anti-PRP (μg/ml) Group N % ≧0.15 LL UL ≧1 LL UL GMC LL UL HibMenCY 2.5/5/5 74 100.0 95.1 100.0 97.3 90.6 99.7 6.441 5.315 7.805 HibMenCY 5/10/10 76 100.0 95.3 100.0 98.7 92.9 100.0 7.324 5.877 9.127 HibMenCY 5/5/5 70 100.0 94.9 100.0 92.9 84.1 97.6 5.577 4.375 7.110Menjugate ™ 74 98.6 92.7 100.0 89.2 79.8 95.2 4.465 3.399 5.865 ActHIB ™74 100.0 95.1 100.0 94.6 86.7 98.5 5.714 4.538 7.195

TABLE 7b SBA-MenC (1/Dil) Group N % ≧1:8 LL UL ≧1:128 LL UL GMT LL ULHib MenCY 2.5/5/5 69 100.0 94.8 100.0 98.6 92.2 100.0 1293.1 1027.71627.1 Hib MenCY 5/10/10 76 100.0 95.3 100.0 97.4 90.8 99.7 1065.6 858.81322.3 Hib MenCY 5/5/5 72 100.0 95.3 100.0 95.8 88.3 99.1 968.4 770.81216.6 Menjugate ™ 74 100.0 95.1 100.0 98.6 92.7 100.0 1931.9 1541.22421.6 ActHIB ™ 76 1.3 0.0 7.1 0.0 0.0 4.7 4.2 3.8 4.5

TABLE 7c Anti-PSC (μg/ml) Group N % ≧0.3 LL UL ≧2 LL UL GMC LL UL HibMenCY 2.5/5/5 63 100.0 94.3 100.0 98.4 91.5 100.0 12.02 9.90 14.59 HibMenCY 5/10/10 65 100.0 94.5 100.0 100.0 94.5 100.0 12.09 10.59 13.81 HibMenCY 5/5/5 61 100.0 94.1 100.0 98.4 91.2 100.0 9.95 8.34 11.87Menjugate ™ 62 100.0 94.2 100.0 100.0 94.2 100.0 15.36 12.67 18.62ActHIB ™ 63 1.6 0.0 8.5 0.0 0.0 5.7 0.15 0.15 0.16

TABLE 7d SBA-MenY (1/Dil) Group N % ≧1:8 LL UL ≧1:128 LL UL GMT LL ULHib MenCY 2.5/5/5 67 98.5 92.0 100.0 95.5 87.5 99.1 843.5 640.1 1111.7Hib MenCY 5/10/10 68 100.0 94.7 100.0 97.1 89.8 99.6 1020.0 790.0 1316.8Hib MenCY 5/5/5 69 98.6 92.2 100.0 89.9 80.2 95.8 741.8 538.0 1022.9Menjugate ™ 68 14.7 7.3 25.4 8.8 3.3 18.2 6.9 5.0 9.5 ActHIB ™ 74 16.28.7 26.6 9.5 3.9 18.5 7.3 5.2 10.1

TABLE 7e Anti-PSY (μg/ml) Group N % ≧0.3 LL UL ≧2 LL UL GMC LL UL HibMenCY 2.5/5/5 67 100.0 94.6 100.0 100.0 94.6 100.0 19.22 15.42 23.95 HibMenCY 5/10/10 70 100.0 94.9 100.0 98.6 92.3 100.0 19.09 15.44 23.59 HibMenCY 5/5/5 72 100.0 95.0 100.0 97.2 90.3 99.7 15.83 12.64 19.82Menjugate ™ 66 3.0 0.4 10.5 0.0 0.0 5.4 0.16 0.15 0.17 ActHIB ™ 69 0.00.0 5.2 0.0 0.0 5.2 0.15 0.15 0.15

Conclusion

The 2.5/5/5 and 5/10/10 formulations resulted in higher titres againstHib, MenC and MenY in terms of immunogenicity and SBA results. Thereforethe inclusion of lower doses of Hib conjugate in a combined conjugatevaccine gave superior results.

Co-administration of Hib-MenCY with Infanrix penta and Prevenar™ gavesatisfactory results

Example 5b Effect of Co-Administration of HibMenCY with Prevenar™ on theResponse to Pneumococcal Polysaccharides

A further aspect of the study of example 3 was to investigate the levelof antibodies raised against the 7 pneumococcal polysaccharides presentin the Prevenar™ vaccine in order to assess the effect ofco-administration of HibMenCY on the antibody titre raised againstpneumococcal polysaccharides.

The GMCs and percentages of subjects with antibodies for the 7pneumococcal serotypes ≧0.05 μg/ml and ≧0.2 μg/ml are shown in Table 8.Except for the 6B serotype, seropositivity rates for the 7vPn componentsranged from 95.5-100% (antibody concentrations ≧0.05 μg/ml) and93.9-100% (antibody concentrations ≧0.2 μg/ml) across groups. For the 6Bserotype, seropositivity rates ranged from 88.4-98.6% (antibodyconcentrations ≧0.05 μg/ml) and 81.2-91.4% (antibody concentrations ≧0.2μg/ml) across groups (ActHIB group: 92.3%≧0.05 μg/ml; 86.2%≧0.2 μg/ml).

TABLE 8a Anti-4 No. in GMC Group group % ≧0.05 μg/ml % ≧0.2 μg/ml(μg/ml) Hib-MenCY 69 100% 100% 2.101 2.5/5/5 Hib-MenCY 70 100% 100%2.049 5/10/10 Hib-MenCY 69 100% 100% 2.023 5/5/5 Menjugate ™ 58  3.4% 1.7% 0.024 ActHib ™ 66 100% 100% 2.062

TABLE 8b Anti-6B No. in GMC Group group % ≧0.05 μg/ml % ≧0.2 μg/ml(μg/ml) Hib-MenCY 68 95.6% 85.3% 1.060 2.5/5/5 Hib-MenCY 70 98.6% 91.4%1.079 5/10/10 Hib-MenCY 69 88.4% 81.2% 0.834 5/5/5 Menjugate ™ 63 4.8%1.6% 0.027 ActHib ™ 65 92.3% 86.2% 0.879

TABLE 8c Anti-9V No. in GMC Group group % ≧0.05 μg/ml % ≧0.2 μg/ml(μg/ml) Hib-MenCY 68 100% 100% 3.102 2.5/5/5 Hib-MenCY 71 98.6%  97.2% 2.363 5/10/10 Hib-MenCY 71 100% 100% 2.823 5/5/5 Menjugate ™ 62  4.8% 1.6% 0.028 ActHib ™ 67 98.5%  98.5%  2.651

TABLE 8d Anti-14 No. in GMC Group group % ≧0.05 μg/ml % ≧0.2 μg/ml(μg/ml) Hib-MenCY 65 100% 98.5% 4.095 2.5/5/5 Hib-MenCY 65 100%  100%5.592 5/10/10 Hib-MenCY 68 100%  100% 4.309 5/5/5 Menjugate ™ 49  49%14.3% 0.062 ActHib ™ 65 100% 98.5% 4.372

TABLE 8e Anti-18C No. in GMC Group group % ≧0.05 μg/ml % ≧0.2 μg/ml(μg/ml) Hib-MenCY 67 98.5% 98.5% 3.518 2.5/5/5 Hib-MenCY 71  100% 98.6%2.969 5/10/10 Hib-MenCY 72  100%  100% 2.936 5/5/5 Menjugate ™ 65  7.7% 3.1% 0.029 ActHib ™ 67 98.5%   97% 3.326

TABLE 8f Anti-19F No. in GMC Group group % ≧0.05 μg/ml % ≧0.2 μg/ml(μg/ml) Hib-MenCY 65 100%  100% 2.303 2.5/5/5 Hib-MenCY 67 98.5%  98.5%1.846 5/10/10 Hib-MenCY 66 100% 100% 2.061 5/5/5 Menjugate ™ 56 12.5%  3.6% 0.030 ActHib ™ 65 100% 96.9% 1.881

TABLE 8g Anti-23F No. in GMC Group group % ≧0.05 μg/ml % ≧0.2 μg/ml(μg/ml) Hib-MenCY 66 98.5%   97% 2.581 2.5/5/5 Hib-MenCY 68 97.1% 94.1%2.112 5/10/10 Hib-MenCY 70 95.7% 95.7% 2.098 5/5/5 Menjugate ™ 59  5.1% 0.0% 0.027 ActHib ™ 66 95.5% 93.9% 1.988

Conclusion

Co-administration of all three formulations of HibMenCY with Prevnar ledto satisfactory immune responses against the seven pneumococcalserotypes. Serotype 6B is a difficult immunogen to raise a responseagainst. In the case of 6B, a higher GMC and percentage of subjectsachieving the two threshold levels was achieved using the lower Hib doseformulations of HibMenC. Therefore the uses of lower dose Hib conjugatevaccines for co-administration with pneumococcal polysaccharideconjugates leads to a better response against the 6B antigen.

Example 6 Phase II Clinical Trial Administering Hib MenCY Concomitantlywith Infanrix Penta According to a 2, 3 and 4 Month Schedule

Study Design:

A Phase II, open (partially double-blind*) randomized controlledmulti-center study with 5 groups receiving a three-dose primary schedulewith vaccines as follows:

Group Hib-MenCY 2.5/5/5: Hib-MenCY (2.5/5/5)+Infanrix™ penta

Group Hib-MenCY 5/10/10: Hib-MenCY (5/10/10)+Infanrix™ penta

Group Hib-MenCY 5/5/5: Hib-MenCY (5/5/5)+Infanrix™ penta

Group Hib-MenC: Hib-MenC (5/5)+Infanrix™ penta

Group Menjugate: Menjugate™**+Infanrix™ hexa (control).

*Hib-MenCY 2.5/5/5, Hib-MenCY 5/10/10 and Hib-MenC were administered ina double-blind manner while the Hib-MenCY 5/5/5 group and the Menjugategroup were open.

**Menjugate™ was the vaccine that was administered to all subjects inthe group. Vaccination at +/−2, 3, 4 months of age (StudyMonth 0, Month1 and Month 2), and blood samples (3.5 ml) from all subjects prior toand one month post primary vaccination (StudyMonth 0 and Month 3).

Study Vaccine, Dose, Mode of Administration, Lot Number:

Three doses injected intramuscularly at one month intervals, atapproximately 2, 3 and 4 months of age as follows:

TABLE 8 Vaccines administered (study and control), group, schedule/siteand dose Vaccine dose Concomitant vaccine Schedule administeredadministered Group (months of age) Site- Left upper thigh Site Rightupper thigh Hib-MenCY 2.5/5/5 2, 3, and 4 Hib (2.5 μg)-MenC-TTDTPa-HBV-IPV (5 μg)-MenY-TT (5 μg) (Infanrix ™ penta) Hib-MenCY 5/10/102, 3, and 4 Hib (5 μg)-MenC-TT DTPa-HBV-IPV (10 μg)-MenY-TT (10 μg)(Infanrix ™ penta) Hib-MenCY 5/5/5 2, 3, and 4 Hib (5 μg)-MenC-TTDTPa-HBV-IPV (5 μg)-MenY-TT (5 μg) (Infanrix ™ penta) Hib-MenC 2, 3, and4 Hib (5 μg)-Men C (5 μg) DTPa-HBV-IPV (Infanrix ™ penta) Menjugate ™ 2,3, and 4 Menjugate ™ DTPa-HBV-IPV/Hib (Infanrix ™hexa)

Immunogenicity:

Measurement of antibody titres/concentrations against each vaccineantigen:

Prior to the first dose (Month 0) and approximately one month after thethird dose (Month 3) in all subjects for: SBA-MenC and SBA-MenY,anti-PSC and anti-PSY, anti-PRP, anti-T, anti-FHA, anti-PRN and anti-PT.Using serum bactericidal activity against N. meningitidis serogroups Cand Y (SBA-MenC and SBA-MenY cut-off: 1:8 and 1:128); ELISA assays withcut-offs: ≧0.3 μg/ml and 2 μg/ml for anti-N. meningitidis serogroups Cand Y polysaccharides (anti-PSC IgG and anti-PSY IgG); ≧0.15 μg/ml and≧1.0 μg/ml for Hib polysaccharide polyribosil-ribitol-phosphate(anti-PRP IgG); 5 EL.U/ml for anti-FHA, anti-PRN, anti-PT; ≧0.1 IU/mlanti-tetanus toxoid (anti-TT). Only at one month after the third dose(Month 3) in all subjects for: anti-D, anti-HBs and anti-polio 1, 2 and3. Using ELISA assays with cut-offs: 0.1 IU/ml for anti-diphtheria(anti-D); ≧10 mlU/ml for antihepatitis B (anti-HBs); andmicroneutralization test cut-off: 1:8 for anti-polio type 1, 2 and 3(anti-polio 1, 2 and 3).

Statistical Methods:

The seroprotection/seropositivity rates and geometric meanconcentrations/titres (GMCs/GMTs) with 95% confidence intervals (95% CI)were computed per group, for SBA-MenC, anti-PSC, SBA-MenY, anti-PSY,anti-PRP, anti-Tetanus, anti-PT, anti-FHA and anti-PRN prior to and onemonth after vaccination; for anti-Diphtheria, anti-HBs, anti-Polio 1,anti-Polio 2 and anti-Polio 3 one month after vaccination. Vaccineresponse (appearance of antibodies in subjects initially seronegative orat least maintenance of antibody concentrations in subjects initiallyseropositive) with 95% CI for anti-PT, anti-PRN and anti-FHA were alsocomputed one month after vaccination. Reverse cumulative curves for eachantibody at Month 3 are also presented. The differences between theHib-MenCY and the Hib-MenC groups, compared with the Menjugate™ controlgroup were evaluated in an exploratory manner for each antibody, exceptfor SBA-MenY and anti-PSY, in terms of (1) the difference between theMenjugate™ group (minus) the Hib-MenCY and Hib-MenC groups for thepercentage of subjects above the specified cut-offs or with a vaccineresponse with their standardized asymptotic 95% CI, (2) the GMC or GMTratios of the Menjugate™ group over the Hib-MenCY and Hib-MenC groupswith their 95% Cl. The same comparisons were done to evaluate thedifference between each pair of Hib-MenCY formulations for anti-PRP,SBA-MenC, anti-PSC, SBA-MenY, anti-PSY and anti-TT antibodies.

The overall incidences of local and general solicited symptoms werecomputed by group according to the type of symptom, their intensity andrelationship to vaccination (as percentages of subjects reportinggeneral, local, and any solicited symptoms within the 8 days followingvaccination and their exact 95% Cl). Incidences of unsolicited symptomswere computed per group. For Grade 3 symptoms, onset hours, medicalattention, duration, relationship to vaccination and outcomes wereprovided. Serious Adverse Events were fully described.

Seroprotection/Seropositivity Rates &GMC/Ts (ATP Cohort forImmunogenicity)

TABLE 9a Anti-PRP (μg/ml) Group N % ≧0.15 LL UL ≧1 LL UL GMC LL UL HibMenCY 2.5/5/5 67 100.0 94.6 100.0 98.5 92.0 100.0 9.01 7.25 11.21 HibMenCY 5/10/10 67 100.0 94.6 100.0 98.5 92.0 100.0 9.49 7.72 11.65 HibMenCY 5/5/5 70 100.0 94.9 100.0 98.6 92.3 100.0 8.08 6.53 9.98 Hib MenC74 100.0 95.1 100.0 98.6 92.7 100.0 10.44 8.49 12.83 Menjugate ™ 71100.0 94.9 100.0 80.3 69.1 88.8 2.60 1.97 3.43

TABLE 9b SBA-MenC (Titre) Group N % ≧1:8 LL UL ≧1:128 LL UL GMT LL ULHib MenCY 2.5/5/5 70 100.0 94.9 100.0 95.7 88.0 99.1 1005.8 773.5 1308.0Hib MenCY 5/10/10 67 100.0 94.6 100.0 94.0 85.4 98.3 1029.8 799.7 1326.0Hib MenCY 5/5/5 71 100.0 94.9 100.0 94.4 86.2 98.4 906.9 691.3 1189.8Hib MenC 74 100.0 95.1 100.0 95.9 88.6 99.2 871.0 677.3 1120.0Menjugate ™ 71 100.0 94.9 100.0 100.0 94.9 100.0 3557.6 2978.8 4248.8

TABLE 9c Anti-PSC (μg/ml) Group N % ≧0.3 LL UL ≧2 LL UL GMC LL UL HibMenCY 2.5/5/5 69 100.0 94.8 100.0 100.0 94.8 100.0 21.70 18.36 25.65 HibMenCY 5/10/10 66 100.0 94.6 100.0 100.0 94.6 100.0 27.26 23.26 31.95 HibMenCY 5/5/5 70 100.0 94.9 100.0 100.0 94.9 100.0 19.02 16.49 21.93 HibMenC 74 100.0 95.1 100.0 100.0 95.1 100.0 21.08 18.24 24.35 Menjugate ™71 100.0 94.9 100.0 100.0 94.9 100.0 38.49 33.64 44.05

TABLE 9d SBA-MenY (Titre) Group N % ≧1:8 LL UL ≧1:128 LL UL GMT LL ULHib MenCY 2.5/5/5 69 97.1 89.9 99.6 92.8 83.9 97.6 470.7 351.1 631.2 HibMenCY 5/10/10 66 97.0 89.5 99.6 86.4 75.7 93.6 437.1 322.0 593.4.8 HibMenCY 5/5/5 71 98.6 92.4 100.0 95.8 88.1 99.1 635.3 501.5 804.8 Hib MenC74 21.6 12.9 32.7 13.5 6.7 23.5 9.3 6.3 13.7 Menjugate ™ 71 19.7 11.230.9 9.9 4.1 19.3 7.5 5.4 10.4

TABLE 9e Anti-PSY (μg/ml) Group N % ≧0.3 LL UL ≧2 LL UL GMC LL UL HibMenCY 2.5/5/5 69 100.0 94.8 100.0 100.0 94.8 100.0 26.86 22.86 31.56 HibMenCY 5/10/10 66 100.0 94.6 100.0 100.0 94.6 100.0 37.02 31.84 43.04 HibMenCY 5/5/5 70 100.0 94.9 100.0 100.0 94.9 100.0 23.57 19.94 27.86 HibMenC 74 8.1 3.0 16.8 4.1 0.8 11.4 0.19 0.15 0.25 Menjugate ™ 71 5.6 1.613.8 1.4 0.0 7.6 0.17 0.15 0.19

TABLE 9e Anti-tetanus (IU/ml) Group N % ≧0.1 LL UL GMC LL UL Hib MenCY2.5/5/5 68 100.0 94.7 100.0 3.06 2.63 3.55 Hib MenCY 5/10/10 67 100.094.6 100.0 3.25 2.88 3.68 Hib MenCY 5/5/5 70 100.0 94.9 100.0 2.97 2.593.41 Hib MenC 74 100.0 95.1 100.0 3.15 2.73 3.64 Menjugate ™ 71 100.094.9 100.0 1.66 1.39 1.97 Group Hib-MenCY 2.5/5/5: Hib-MenCY (2.5/5/5) +Infanrix ™ penta Group Hib-MenCY 5/10/10: Hib-MenCY (5/10/10) +Infanrix ™ penta Group Hib-MenCY 5/5/5: Hib-MenCY (5/5/5) + Infanrix ™penta Group Hib-MenC: Hib-Men (5/5) + Infanrix ™ hex Group Menjugate:Menjugate ™ + Infanrix ™ penta N = number of subjects with availableresults. % = percentage of subjects with concentration/titre within thespecified range GMC/T: geometric mean concentration/titre 95% CI = 95%confidence interval; LL = Lower Limit; UL = Upper Limit

CONCLUSION

The immune responses against Hib and MenC were superior using the twoformulations with reduced doses of Hib. For MenY, an improved SBAresponse was seen using the 2.5/5/5 and 5/10/10 formulations compared tothe 5/5/5 formulation.

The invention claimed is:
 1. A process for making an immunogeniccomposition, said immunogenic composition comprising: a Haemophilusinfluenza type b (Hib) saccharide conjugate; an N. meningitidis capsularsaccharide conjugate derived from a strain selected from the groupconsisting of serogroups A, B, C, W135 and Y; whole cell pertussis; andhepatitis B surface antigen; the method comprising; mixing the Hibsaccharide conjugate with at least one N meningitidis capsularsaccharide conjugate and with the whole cell pertussis and hepatitis Bsurface antigen to form a composition wherein the saccharide dose of theHib saccharide conjugate is less than 5 ug and the saccharide does ofthe N. meningitidis capsular saccharide conjugate is less than 8 μg, andwherein the Geometric Mean Concentration (GMC) for whole cell pertussisor hepatitis B achieved with said immunogenic composition is higher thanthe GSM achieved after immunisation with said immunogenic compositioncontaining a 5 μg saccharide dose of Hib saccharide conjugate and a 8 μgdose of a N. meningitidis capsular saccharide conjugate.